The interaction of specific DNA-binding proteins with their recognition sites in the eukaryotic chromosome is central to the mechanisms of gene regulation that occur in these cells. The techniques used to study these interactions rely primarily on methodology designed to detect preferential binding between proteins at various levels of purity from broken cell preparations and purified DNA sequences. It is often assumed that if a high affinity binding protein is present in a given cell type, that protein will interact with its recognition sequence. We recently observed (see Project Z01CP04986-10 LEC) that two tight- binding transcription factors are excluded from MMTV chromatin in vivo. These findings indicate the importance of techniques that permit an unambiguous determination as to when a protein occupies (or is excluded from) its recognition site in vivo. We have undertaken to apply the technique of UV DNA-protein cross-linking to this problem. Several advantages would accrue from the successful development of this application. Utilizing amplified minichromosomes, based on the bovine papilloma virus (BPV) vector (see Project Z01CP05450-03 LEC), we have detected specific cross-links between minichromosome DNA two types of proteins, RNA polymerase II and histones. Using the MMTV hormone-inducible promoter on BPV minichromosomes, the interactions of both proteins are modified by steroid induction. Attempts are underway to extend this technology to the use of very short (40 nanosecond) irradiations, which would offer a new and powerful approach to the study of transcriptional regulation.